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ABSTRACT Plants monitor seasonal cues to optimize reproductive success by tuning onset of reproduction and inflorescence architecture. TERMINAL FLOWER 1 (TFL1) and FLOWERING LOCUS T (FT) and

their orthologs antagonistically regulate these life history traits, yet their mechanism of action, antagonism and targets remain poorly understood. Here, we show that TFL1 is recruited to

thousands of loci by the bZIP transcription factor FD. We identify the master regulator of floral fate, _LEAFY_ (_LFY_) as a target under dual opposite regulation by TFL1 and FT and uncover

a pivotal role of FT in promoting flower fate via _LFY_ upregulation. We provide evidence that the antagonism between FT and TFL1 relies on competition for chromatin-bound FD at shared

target loci. Direct TFL1-FD regulated target genes identify this complex as a hub for repressing both master regulators of reproductive development and endogenous signalling pathways. Our

data provide mechanistic insight into how TFL1-FD sculpt inflorescence architecture, a trait important for reproductive success, plant architecture and yield. SIMILAR CONTENT BEING VIEWED BY

OTHERS LEAFY IS A PIONEER TRANSCRIPTION FACTOR AND LICENSES CELL REPROGRAMMING TO FLORAL FATE Article Open access 27 January 2021 TWO FLORIGENS AND A FLORIGEN-LIKE PROTEIN FORM A TRIPLE

REGULATORY MODULE AT THE SHOOT APICAL MERISTEM TO PROMOTE REPRODUCTIVE TRANSITIONS IN RICE Article 27 March 2023 THE F-BOX PROTEIN UFO CONTROLS FLOWER DEVELOPMENT BY REDIRECTING THE MASTER

TRANSCRIPTION FACTOR LEAFY TO NEW _CIS_-ELEMENTS Article 02 February 2023 INTRODUCTION Of particular importance for reproductive success of flowering plants is optimal timing of onset of

reproductive development and of the transition from branch to floral fate in the inflorescence in response to seasonal cues1,2,3,4,5. For example, in plants that flower only once, like

_Arabidopsis_ and most crops, an early switch to flower formation allows rapid completion of the life-cycle and is beneficial in a short growing season5,6,7. At the same time early onset of

flower formation reduces seed set and yield since flowers form in lieu of branches, which support production of more flowers per plant5,6,7,8. By contrast, delaying flower formation

increases branching and total flower number, but prolongs time to seed set5,6,7,8. Key regulators of seasonal control of onset of reproductive development and of the switch from branch to

floral fate in primordia of the inflorescence are members of the phosphatidylethanolamine-binding protein (PEBP) family of proteins5,6,9,10. Among these, FT promotes onset of the

reproductive phase and flower formation (determinacy), while TFL1 promotes vegetative development and branch fate (indeterminacy)9,11,12,13. _Arabidopsis_ flowers in the spring and FT

accumulates when the daylength exceeds a critical threshold, while TFL1 is present in both short-day and long-day conditions2,3,14. FT and TFL1 are small mobile proteins, which have been

implicated in transcriptional regulation but do not have DNA-binding domains14,15,16,17,18. Biochemical and genetic studies showed that FT physically interacts with the bZIP transcription

factor FD via 14-3-3 proteins and similar interactions have recently been described for TFL119,20,21,22. Indeed, despite their antagonistic roles, TFL1 and FT are distinguished by only a

small number of nonconservative amino acid changes11,12,23,24. FT can be converted into TFL1 and vice versa by a single amino acid substitution and such mutations have been selected for

during crop domestication23,24,25,26. Accumulating evidence suggests that FT acts as a transcriptional co-activator, while TFL1 may either prevent FT activity or act as a co-repressor23,27.

However, non-nuclear roles have also been described for both TFL1 and FT28,29. A key unanswered question is how the florigens modulate plant form—what are the downstream processes they set

in motion and what is molecular basis for their antagonism? Here we show that TFL1 is recruited to target loci by the bZIP transcription factor FD. We identify the master regulator of floral

fate, LEAFY, as a target under dual opposite regulation by TFL1 and FT and uncover a prominent role for FT in LFY upregulation. We find that the antagonism between TFL1 and FT relies on

competition for access to chromatin bound FD at the _LFY_ locus and other shared targets. Finally, we identify hundreds of TFL1–FD regulated genes linking this complex not only to repression

of master regulators of floral fate, but also of diverse endogenous signalling pathways. The combined data reveals how TFL1 and FT tune inflorescence architecture in response to seasonal

cues by altering transcriptional programs that direct primordium fate in the inflorescence. RESULT TFL1 IS RECRUITED TO THOUSANDS OF LOCI BY THE BZIP TRANSCRIPTION FACTOR FD Mechanistic

insight into TFL1 activity has been hampered by low protein abundance. To overcome this limitation and to test the role of TFL1 in the nucleus, we first generated a biologically active,

genomic GFP-tagged version of TFL1 (gTFL1-GFP _tfl1-1_) (Supplementary Fig. 1a–c) and identified a developmental stage and tissue where TFL1 accumulates. TFL1 protein strongly accumulated in

branch meristems in the axils of cauline leaves in 42-day-old short-day grown plants just prior to the switch to flower formation (Fig. 1a). To conduct TFL1 chromatin immunoprecipitation

followed by sequencing (ChIP-seq), we next isolated shoot apices at this stage for anti-GFP immunoprecipitation. Because TFL1 is present in very few cells and binds chromatin indirectly, we

combined eight individual ChIP reactions per replicate to enhance detection. We conducted FD ChIP-seq in analogous fashion using a published, biologically active, genomic fusion protein

(gFD-GUS _fd-1_)20 (Supplementary Fig. 1d). This approach yielded high-quality ChIP-seq data in both cases (Supplementary Figs. 2 and 3a). In total, we identified 3308 and 4422 significant

TFL1 and FD peaks (MACS2 summit qval ≤ 10−10), respectively (Fig. 1b). The TFL1 peaks significantly overlapped with the FD peaks (72% overlap, _p_ val < 10−300, hypergeometric test; Fig. 

1b–d). De novo motif analysis of ChIP peak summits identified the G-box _cis_ motif, a known FD-binding site30, as most significantly enriched (_p_ val < 10−470) and frequently present

(>84%) under TFL1 bound and TFL1/FD co-bound peaks (Fig. 1e and Supplementary Fig. 2). To test whether TFL1 chromatin occupancy is dependent on the presence of FD, we also performed TFL1

ChIP-seq in the _fd-1_ null mutant. TFL1 chromatin occupancy was strongly reduced in _fd-1_ (Fig. 1c, d). Our data point to a prominent nuclear role for TFL1 and show that FD recruits TFL1

to the chromatin of target loci. Annotating FD and TFL1 peaks to genes identified 2699 joint TFL1 and FD targets. Gene Ontology (GO) term enrichment analysis implicates these targets in

abiotic and endogenous stimulus response and reproductive development (Supplementary Table 1). TFL1 and FD peaks were present at loci that promote onset of the reproductive phase in response

to inductive photoperiod2,3,31 like _GIGANTEA_ (_GI_), _CONSTANS_ (_CO_), and _SUPPRESSOR OF CONSTANS 1_ (_SOC1_) and at loci that promote floral fate31,32 such as _LFY_, _APETALA1_

(_AP1_), and _FRUITFULL_ (_FUL_) (Fig. 1f). Identification of these TFL1 and FD co-bound targets fits with the known biological role of TFL1 as a suppressor of onset of reproduction and of

flower fate and the proposed molecular function of TFL1 in opposing gene activation11,12,13,27. LEAFY IS UNDER DUAL OPPOSITE REGULATION BY TFL1/FD AND FT/FD We selected the _LEAFY_ (_LFY_)

gene, which encodes a master regulator of flower fate33,34, to further probe the molecular mechanism of action of TFL1. While TFL1 promotes branch fate, LFY promotes flower fate in primordia

(Supplementary Fig. 4a–f)13,33,34,35. Using independent biological replicates, we confirmed FD-mediated TFL1 binding to _LFY_ by ChIP-qPCR (Supplementary Fig. 4g, h). To test whether _LFY_

expression is rapidly repressed by the TFL1–FD complex, we generated transgenic plants expressing a steroid inducible version of TFL1 (TFL1ER; Supplementary Fig. 5). A single steroid

treatment reduced _LFY_ levels by 50% after 4 h (Supplementary Fig. 4i). The combined data suggest that the TFL1–FD complex directly represses _LFY_. To better understand TFL1 recruitment to

the _LFY_ locus, we identified the genomic region sufficient and the _cis_ motifs necessary for TFL1 association with the _LFY_ locus. TFL1 and FD peak summits located to the second exon of

_LFY_ (Fig. 1f and Supplementary Fig. 4g, h) and _LFY_ reporters that lacked the second exon were not repressed in response to TFL1 overexpression (Supplementary Fig. 6a, b). Exonic

transcription factor-binding sites, although rare, are found in both animals and plants, and frequently link to developmental regulation36,37. To test whether LFY exon 2 (e2) alone is

sufficient to recruit TFL1–FD, we transformed gTFL1-GFP _tfl1-_1 plants with a T-DNA containing only _LFY_ e2. We detected strong TFL1 recruitment to the introduced copy of e2, using primer

sets that specifically amplify the transgene borne exon (Fig. 2a). Next we identified three putative bZIP-binding sites in the second exon of _LFY;_ These include an evolutionarily conserved

G-box and two partially conserved C-boxes (Fig. 2b). When we transformed gTFL1-GFP _tfl1-_1 with a version of LFY e2 in which the three bZIP binding were mutated (e2m3), we were unable to

detect TFL1 binding to the introduced copy of e2m3 (Fig. 2a). The three bZIP-binding sites in the second exon of _LFY_ are thus necessary for TFL1 recruitment. Similar results were obtained

when we tested TFL1 recruitment to e2 and e2m3 via FD in yeast (Supplementary Fig. 6c). Having identified the _cis_ motifs necessary for TFL1 recruitment to _LFY_, we next probed their

contribution to spatiotemporal _LFY_ accumulation. _LFY_ reporters that contain e2 (pLFYi2-GUS, Supplementary Fig. 6a) and a genomic _LFY_ reporter (gLFY-GUS, Fig. 2b) recapitulated

endogenous LFY expression (Fig. 2c, Supplementary Fig. 6d)34,35. Mutating the three bZIP-binding sites in pLFYi2-GUS or gLFY-GUS caused ectopic reporter expression in the centre of the

inflorescence shoot apex (Fig. 2c, Supplementary Fig. 6d). This is the precise region where TFL1 protein accumulates during reproductive development (Fig. 2c)14. Indeed, _LFY_ is known to be

ectopically expressed in the inflorescence shoot apex of _tfl1_ mutants during reproductive development13. Thus, TFL1–FD binding to the bZIP motifs of e2 of LFY is required to prevent

ectopic _LFY_ accumulation in the centre of the shoot apex. Surprisingly, the bZIP-binding site mutations in the second exon of _LFY_ in addition strongly reduced reporter expression in

incipient and young flower primordia (Fig. 2c, Supplementary Fig. 6d). This suggests that the bZIP motifs may be required for _LFY_ upregulation in these flower primordia, perhaps via FT.

Based on prior studies31,38,39,40,41, _LFY_ was not thought to be an immediate early FT target. Because constitutive mutants that delay onset of the reproductive phase, like _ft_, indirectly

delay the switch to flower formation42 we wished to deplete FT specifically during the reproductive phase to test whether FT promotes _LFY_ expression. Towards this end, we used a minimal

_FT_ promoter (p4kbFT) that is active in parts of leaves and stems in long-day grown plants only after day 1243. We fused p4kbFT to a previously characterized FT-specific artificial

microRNA44. In the resulting conditional _ft_ mutant (p4kbFT:amiRFT), onset of reproduction was not delayed (Fig. 3a, Supplementary Fig. 7). However, p4kbFT:amiRFT plants displayed a

significantly delay in onset of flower formation and failed to upregulate _LFY_ expression (Fig. 3a and Supplementary Fig. 7). In a parallel approach to test the role of _FT_ in LFY

induction, we induced endogenous _FT_ expression by treating 42-day-old short-day grown plants with a single far-red-enriched long-day photoperiod (FRP). Far-red light enhances _FT_

induction by photoperiod (Supplementary Fig. 8a, b)45,46. In addition, FRP triggered significant _LFY_ induction, which was dependent on the presence of FT (Supplementary Fig. 8c, d). A

single FRP treatment also induced the gLFY-GUS reporter, but only if the bZIP-binding sites in e2 were intact (Fig. 3b). Finally, we probed for rapid _LFY_ induction by FT after generating

an estradiol inducible version of FT (35S:FT-HAER) (Supplementary Fig. 9a–c). A single steroid treatment triggered significant _LFY_ induction after 4 h (Supplementary Fig. 9d). After

crossing FT-HAER to LFY reporters containing (pLFYi2:GUS) or lacking (pLFYi2m3:GUS) the bZIP-binding sites in e2, we tested reporter activity in response to steroid activation. GUS

upregulation was similar to that of endogenous _LFY_ when the bZIP-binding sites were present (Supplementary Fig. 9e). By contrast, GUS expression was not upregulated in the pLFYi2m3:GUS

FT-HAER plants after steroid induction (Supplementary Fig. 9e). The combined loss-of-function, photoinduction and gain-of-function data indicate that FT–FD directly activates _LFY_

expression via bZIP-binding sites in the second exon. These findings prompted us to assess the biological importance of the _LFY_ bZIP-binding sites for inflorescence architecture. While a

genomic GFP-tagged _LFY_ construct (gGLFY) fully rescued the _lfy-1_ null mutant (in 24 out of 25 independent transgenic lines), a construct which preserves LFY protein sequence but has

mutated bZIP-binding sites (gGLFYm3) yielded only partial rescue (in 15 out of 15 independent transgenic lines) (Fig. 3c, d and Supplementary Fig. 10). In the gGLFYm3 _lfy-1_ plants onset of

flower formation was significantly delayed, leading to formation of many more branches, and _LFY_ accumulation was strongly reduced (Fig. 3c, d and Supplementary Fig. 10). The dramatic

reduction of _LFY_ accumulation is striking given the many additional positive inputs into _LFY_ upregulation previously identified38,40,47,48. Our combined data uncover a pivotal role of FT

in _LFY_ upregulation and reveal that FT promotes flower formation via _LFY_. We note that _LFY_ accumulation in the centre of the gGLFYm3 _lfy-1_ shoot apex was much lower than that

observed in _tfl1_ mutants and that gGLFYm3 _lfy-1_ did not exhibit the terminal flower phenotype typical of _tfl1_ (Supplementary Fig. 10)13. This is expected since the bZIP mutations at

the _LFY_ locus prevent access of both TFL1 and of activating PEBP family members FT and the closely related TWIN SISTER OF FT (TSF). Indeed, it has been shown that the terminal flower

phenotype of _tfl1_ is suppressed in _tfl1 ft tsf_ triple mutants49. FT COMPETES TFL1 FROM FD BOUND AT SHARED TARGET LOCI Having identified _LFY_ as a target under dual opposite regulation

by TFL1 and FT, we next investigated the mechanism underlying the TFL1–FT antagonism at this locus. To test for possible competition between FT and TFL1 at the chromatin, we conducted

anti-HA ChIP-qPCR in 42-day-old short-day grown FT-HAER gTFL1-GFP plants four hours after mock or steroid application. Estradiol induction led to rapid recruitment of FT-HA to the second

exon of _LFY_, the region occupied by FD and TFL1 (compare Fig. 4a, b to Supplementary Fig. 4h). Anti-GFP ChIP-qPCR performed on the same sample uncovered a concomitant reduction in TFL1

occupancy (Fig. 4a, b). We next asked whether upregulation of endogenous FT also triggers reduced TFL1 occupancy at the _LFY_ locus. Towards this end, we treated plants with a single FRP to

upregulate FT (Supplementary Fig. 8b). The single FRP likewise significantly reduced TFL1 occupancy at the _LFY_ chromatin (Fig. 4c). By contrast, photoinduction of FT did not alter FD

occupancy (Fig. 4d). To probe whether FT is recruited to the second exon of LFY via the bZIP-binding sites, we transformed FT-HAER with either a wild-type version of e2 or a bZIP-binding

site mutated version thereof (e2m3). After steroid induction, we used transgene-specific primers to monitor FT binding to the two versions of _LFY_ e2 by ChIP-qPCR. As described above for

TFL1 (Fig. 2a), FT was recruited to _LFY_ e2 alone (Fig. 4e). In addition, FT recruitment to the introduced copy of _LFY_ e2 was abolished when the three bZIP-binding sites were mutated

(Fig. 4e). Our combined data suggest that FT competes TFL1 from FD bound at exonic bZIP motifs at the _LFY_ locus. The loss of TFL1 from the LFY locus via competition by FT is further

supported by the finding that neither steroid nor FRP induction of FT reduced _TFL1_ mRNA accumulation (Supplementary Figs. 8c and 9d). Competition of TFL1 from FD by FT is not limited to 

the _LFY_ locus. We tested whether FT induction by FRP competes TFL1 from the other direct TFL1–FD target loci we identified (Fig. 1f). FRP treatment reduced TFL1 occupancy at all loci

tested (Fig. 4f). To confirm that FT indeed occupies the TFL1–FD bound sites at these loci, we also conducted ChIP-qPCR in FT-HAER after steroid induction. In the estradiol treated samples,

we saw significant FT recruitment to the TFL1–FD bound regions at all loci tested (Fig. 4g). We conclude that the antagonism between FT and TFL119,21,27 relies on competition for FD bound at

the chromatin of shared target loci (Fig. 4h). DIRECT TFL1–FD REPRESSED GENES PROMOTE ONSET OF FLOWER FORMATION AND ENDOGENOUS SIGNALLING Our findings place florigens directly upstream of

LFY, yet prior genetic data suggest that florigens act both upstream of and in parallel with LFY39,50. To gain insight into additional gene expression programs repressed by the TFL1–FD

complex, we next conducted RNA-seq with and without FRP treatment. We isolated inflorescences with associated primordia from 42-day-old short-day-grown _ft_ mutant, wild-type and _tfl1_

mutant plants and identified the significant gene expression changes in each genotype relative to untreated siblings. On the basis of Principle Component Analysis (PCA) and replicate

analysis, RNA-seq quality was high (Supplementary Fig. 11). We next defined genes directly repressed by TFL1–FD. Towards this end, we focussed on TFL1–FD complex bound loci that exhibit

FT-dependent de-repression upon photoinduction. Six-hundred four TFL1–FD bound genes were significantly (DESeq2 adjusted _p_ < 0.005) de-repressed upon FRP treatment in the wild-type or

in _tfl1_ mutants but not in _ft_ mutants (Fig. 5a). GO term enrichment linked the TFL1–FD repressed genes to reproductive development and to response to endogenous and abiotic signals (Fig.

 5b). K-means clustering of the 604 genes identified three main patterns of gene expression. Genes encoding promoters of floral fate32 (_LFY_, _AP1_, _FUL_ and _LMI2_) clustered together

(cluster III in Fig. 5c) and displayed stronger upregulation in _tfl1_ mutants than in the wild type. This pattern of de-repression was confirmed for all four loci using independent

biological samples and qRT-PCR (Supplementary Fig. 12a). _SOC1_ clusters with these genes, but was not included in further analyses because it was weakly, but significantly, de-repressed in

_ft_ mutants (Fig. 5c, Supplementary Data 1 and Supplementary Fig. 12b). By contrast, _CO_ and _GI_, which promote cessation of vegetative development2,3, were more strongly upregulated in

the wild-type than in _tfl1_ mutants (cluster I in Fig. 5c), perhaps because these genes are already partially de-repressed in _tfl1_ mutants in the absence of FRP treatment. Indeed, like

RNA-seq, qRT-PCR of independent biological replicates revealed higher accumulation of _GI_ and _CO_ in untreated _tfl1_ mutant compared to wild-type plants (Fig. 5c, Supplementary Data 1 and

Supplementary Fig. 12b). Cluster II genes are only upregulated in the wild type and may represent genes that are transiently de-repressed. Our data identify the TFL-FD complex as a hub for

repression of key regulators of the onset of reproductive development and of the switch to flower fate (Fig. 5c) Consistent with the GO-term enrichment analysis (Fig. 5b), combined ChIP-seq

and RNA-seq analysis additionally identified components of endogenous stimulus response. We identified genes linked to sugar signalling (trehalose-6-phosphate) and hormonal signalling and

response (abscisic acid, cytokinin, brassinosteroid, auxin and strigolactone) as direct TFL1–FD complex repressed targets (Fig. 5c). Using qRT-PCR and independent biological samples, we

confirmed FT-dependent de-repression of members of these pathways by FRP photoinduction (Fig. 5c). Several of the identified pathways link to repression of branching or to promotion of onset

of flower formation in the inflorescence. For example, we identified four trehalose-6-phosphate phosphatases (_TPPH_, _TPPJ_, _TPPG_ and _TPPE_) as direct TFL1–FD complex repressed targets;

TPPs were recently shown to repress branching in the maize inflorescence51. In addition, auxin and the auxin-activated transcription factor _MONOPTEROS_ (_MP_) were direct TFL1–FD complex

repressed targets (Fig. 5c). MP promotes the switch to floral fate in _Arabidopsis_52 and the tomato ortholog of TFL1 executes its role in inflorescence architecture at least in part by

modulating auxin flux and response53. Finally, we identified key components of the brassinosteroid pathway including the _ BRI1_ receptor and the bHLH transcription factor _BIM1_54,55 as

direct TFL1–FD complex repressed targets. Brassinosteroid signalling represses inflorescence branching in Setaria56. The combined data implicate TFL1–FD in direct repression of genes that

promote floral fate or repress branch fate, consistent with the role of TFL1 in promoting branch formation. We also identified the cytokinin activating enzyme _LOG5_57, abscisic acid

biosynthesis (_ABA1_) and response regulators (_ABI5_, _ABF4_, _APF2_)58,59, and components of strigolactone signalling, _SMXL6_ and _SMXL8_60,61, as direct TFL1–FD complex repressed

targets. To gain further insight into the role of the TFL1–FD complex in hormone signalling, we next assessed indirect, downstream, gene expression changes triggered by FRP treatment. In

particular, we identified genes not bound by TFL1 or FD that were significantly differentially expressed (DESeq2 adjusted _p_ < 0.005) in the wild type and in _tfl1_, but not in _ft_. The

identified indirect targets provide a ‘molecular phenotype’ that is consistent with de-repression of the auxin, brassinosteroid and cytokinin hormone pathways upon FRP treatment in the

wild-type and in _tfl1_ mutants (Fig. 5c). By contrast, the abscisic acid signalling pathway signature was more complex (Fig. 5c). Our combined data uncover a prominent role for the TFL1–FD

complex in regulation of endogenous signalling. It is conceivable that components of some of the identified TFL1–FD dependant pathways (strigolactone, cytokinin, auxin, abscisic acid as well

as sugar signalling) may modulate additional aspects of the inflorescence architecture, such as branch outgrowth62,63,64. Support for this hypothesis comes from our phenotypic analyses. We

examined the effect of a single FRP on inflorescence architecture in the _ft_ mutant, the wild-type and the _tfl1_ mutant. Photoperiod induction triggered a reduction in the number of

branches, but not cauline leaves, formed in the wild type and more strongly, in _tfl1_ (Supplementary Fig. 13a–j). This suggests that branch meristems adopt floral fate upon stimulus

perception65. _tfl1_ mutants also formed fewer branches than the wild type in the absence of photoperiod. These phenotypes are consistent with the observed gene expression changes (Fig. 5c).

In addition, FRP triggered a significant increase in inflorescence branch outgrowth in both wild-type and _tfl1_ plants (Supplementary Fig. 13k). FRP had no phenotypic effect in _ft_

mutants. Our combined data suggest that florigens tune plant form to the environment by controlling expression of master developmental regulators and endogenous signalling pathway

components. These developmental changes likely require large-scale transcriptional reprograming in the context of chromatin. Consistently, we identified transcriptional co-regulators and

chromatin regulators among the direct TFL1–FD repressed targets (Supplementary Fig. 14a, b). DISCUSSION Here we identify _LFY_, a master regulator of flower fate33,34, as a target under dual

opposite transcriptional regulation by TFL1 and FT and demonstrate that FT activation of _LFY_ expression is critical to promote floral fate. We provide a molecular framework for the

antagonistic roles5,27 of FT and TFL1 that relies on competition for bZIP transcription factor mediated access to binding sites at regulatory regions of shared target loci. Additional

support for this mechanism comes from recent in vitro studies21. Our data suggest that TFL1 may not simply prevent access of the FT co-activator to the chromatin23 but may be an active

repressor, as mutating bZIP-binding sites results in _LFY_ de-repression specifically in the TFL1 expression domain. The identity of the transcription factors that activate _LFY_ in the

centre of the inflorescence shoot apex in the absence of PEBP/FD binding to _LFY_ is not known. Our identification of FT recruiting motifs in the second exon of _LFY_ fits with prior data

demonstrating that the 2.3 kb upstream intergenic ‘_LFY_ promoter’ is unresponsive to FT38. This upstream regulatory region drives reporter expression in similar domains as endogenous

_LFY_66. The requirement of the bZIP motifs for _LFY_ upregulation in the context of the genomic construct, which contains the 2.3 kb ‘_LFY_ promoter’, suggests the presence of repressive

regulatory elements in the genic region of _LFY_. We identify hundreds of TFL1–FD repressed genes many of which, based on our computational analyses of recently published FD and TFL1

ChIP-seq datasets17,30, are also immediate early gTFL1 or gFD targets in long-day conditions. Eighty two percent of our 604 high-confidence TFL1–FD repressed genes are present in at least

one of the long-day ChIP-seq datasets (Supplementary Fig. 3b). The 604 direct TFL1–FD repressed genes include key regulators of onset of the reproductive phase and of floral fate. Of note,

TFL1 opposes not only LFY, but also LFY targets, such as _LMI2_ and _AP1_67,68. This is consistent with prior genetic investigations that place TFL1 both upstream of LFY and as a modulator

of plant response to LFY50. Finally, we link the TFL1–FD complex to repression of diverse endogenous signalling pathways including sugar and hormonal signals. Several of these pathways have

been shown to impact the switch from branch or flower fate in other plant species51,53,56. The combined data point to an important role of the hormonal environment for the switch from branch

to flower fate in primordia of the inflorescence. Our findings also set the stage for elucidating communalities as well as differences between florigen regulated cell fate reprogramming

during flower initiation and other developmental pathways under seasonal control by florigens such as tuberization, bulb formation and seed dormancy69,70,71,72. Changes in the relative

balance of activating and repressive PEBP family members occurred during domestication of diverse crop species to give rise to desirable traits like everbearing and compact growth

habits5,8,69,73. Thus, mechanistic insight into the antagonism and identification of the targets of PEBPs will benefit traditional or genome editing-based crop improvement. It should further

facilitate elucidation that how PEBP protein act as co-activators or co-repressors in the nucleus. METHODS PLANT MATERIALS _Arabidopsis_ ecotype Columbia plants were grown in soil at 22 °C

in long-day photoperiod (LD, 16 h light/8 h dark, 100 µmol/m2 s) or short-day photoperiod (SD, 8 h light/16 h dark, 120 µmol/m2 s). gFD-GUS20, _fd-1_ null mutants74, _tfl1-14_ hypomorph

mutant27,75, _tfl1-1_ severe mutant27,75,76, _lfy-1_ null mutant33,77,78, _ft-10_ null mutant79, 35S:LFY68 and 35S:TFL1 (ref. 35) were previously described. 35S:LFY (Landsberg _erecta)_ was

introgressed into the Columbia background through backcrossing. _gFD-GUS_80 was crossed into the _fd-1_ null mutant background. CONSTRUCTS FOR TRANSGENIC PLANTS For _gTFL1-GFP_, GFP followed

by a peptide linker (GGGLQ) was fused to an 8.4 kb BamHI (NEB, R0136S) genomic fragment from lambda TFG4 (ref. 81). This fragment was introduced into the binary vector _pCGN1547_ (ref. 82).

For _TFL1__ER_ and _FT_-_HA__ER_, _TFL1_ and _FT_ were PCR amplified from cDNA; in the case of FT, the 3′ primer contained three times Hemagglutinin (HA) plus a stop codon. PCR products

were cloned into _pENTRD-TOPO_ (Invitrogen, K243520) and shuffled into _pMDC7_ (ref. 83) by LR reaction (Invitrogen, 11791-020). For _LFY-GUS_ reporters, the bacterial beta-glucuronidase

(_GUS_) gene from the _pGWB3_ (ref. 84) binary vector was fused with _pENTRD_-_TOPO_ vector containing the 2290-bp _LFY_ promoter66 alone (pLFY:_GUS_), the _LFY_ promoter and _LFY_ genic

region up to and including the first intron (pLFYi1:GUS), or the _LFY_ promoter and _LFY_ genic region up to and including the second _LFY_ intron (_pLFYi2_:GUS) by LR reaction. bZIP-binding

site mutations in _LFY_ e2 (_pLFYi2m3_:GUS_)_ were generated by Ω-PCR85. _gGLFY_ was constructed by PCR amplifying a 4929-bp genomic _LFY_ fragment (_gLFY_), including the 2290-bp _LFY_

promoter, from genomic DNA followed by cloning into the KpnI-HF (NEB, R3124S) and NotI-HF (NEB, R3189S) digested _pENTR3C_ vector by Gibson Assembly. Next, GFP was inserted at position + 94 

bp, as previously described for _pLFY:GLFY_42,80, by Ω-PCR85. bZIP-binding site mutations were introduced into _pENTR3C_-_gGLFY_ by Ω-PCR to generate _gGLFYm3_. Both constructs were shuffled

into _pMCS:GW_86 using LR reaction. To create _gLFY_:_GUS_, the 4929-bp genomic _LFY_ clone minus the stop codon and the GUS fragment were PCR amplified and inserted into linearized

_pENTR3C_ by Gibson Assembly. For _gLFYm3_:_GUS_, bZIP-binding site mutations were introduced into _pENTR3C-gLFY:GUS_ by Ω-PCR. To test recruitment of TFL1 and FT to e2 of _LFY_, wild-type

(e2) or bZIP-binding site mutated exon 2 (e2m3) were PCR amplified and cloned into _pGWB3_ (ref. 84). For test of recruitment, LFY e2 and e2m3 were amplified by forward

(5′-caccAACAGCAGCAGAGACGGAGAAAGAA-3′) and reverse (5′-TCGTACAAGTGGAACAGATAATC-3′) primers and cloned into pGWB3 binary vectors, which were transformed into gTFL1-GFP and 35S:FT-HAER. For

_pFT4kb:amiRFT_, a 3994-bp truncated _FT_ promoter87 was PCR amplified from genomic DNA as was the published _amiRFT_44 from _pRS300_ (ref. 88). The _amiRFT_ fragment was introduced into

EcoRI-HF (NEB, R3101S) digested _pENTR3C_ (Thermo Fisher Scientific, A10464) by Gibson Assembly (NEB, E5510S) and shuffled into binary vector _pMCS:GW_86 using LR reaction, which resulted in

pMCS:amiRFT. The previously described 3994-bp _FT_ promoter was inserted to XhoI (NEB, R0146S) digested pMCS:amiRFT by Gibson Assembly. Genomic DNA was extracted using the GenElute Plant

Genomic DNA Miniprep Kit (Sigma-Aldrich, G2N70). Primer sequences are listed in Supplementary Table 2. All constructs were sequence verified prior to transformation into plants with

Agrobacterium strain GV3101 by floral dip89. Plant lines generated are listed in Supplementary Table 3. IMAGING Images were taken with a Canon EOS Rebel T5 camera for plant phenotypes and

yeast one-hybrid assays, or with a stereo microscope (Olympus SZX12) equipped with a colour camera (Olympus LC30) for GUS images and inflorescence phenotypes. For GFP images, a Leica TCS SP8

Multiphoton Confocal with a 20× objective was used with a 488 nm excitation laser and emission spectrum between 520 and 550 nm (GFP) or 650–700 nm (chlorophyll autofluorescence) using

standard imaging techniques90,91. For _tfl1-1_ gTFL1-GFP in short-day photoperiod, shoot apices were sectioned longitudinally on an oscillating tissue slicer (Electron Microscopy Sciences,

OTS-4000) after embedding in 5% Agar (Fisher Scientific, DF0812-07-1). PLANT TREATMENT AND GENE EXPRESSION ANALYSIS For test of gene expression, 16-day-old TFL1ER or 12-day-old FT-HAER

plants grown in LD were induced by a single spray application of 10 μmol beta-estradiol (Sigma-Aldrich, 8875-250MG) dissolved in DMSO (Fisher Scientific, BP231-1L) and 0.015% Silwet L-77

(PlantMedia, 30630216-3). Mock solution consisted of 0.1% DMSO and 0.015% Silwet. To probe FT recruitment to and TFL1 occupancy at the _LFY_ locus, 42-day-old FT-HAER gTFL1-GFP plants grown

in SD were treated by a single spray application of 10 μmol beta-estradiol or mock solution. In all cases, tissues were harvested 4 h after treatment. To test for gain-of-function phenotypes

in long-day photoperiod, FT-HAER, TFL1ER and FT-HAER gTFL1-GFP plants were treated with 10 μmol beta-estradiol or mock solution from 5-day onwards every other day until bolting. FRP was

applied at the end of the short day (ZT8) for 24 h using a Percival Scientific E30LED45 with red (660 nm) to far-red (730 nm) ratio = 0.5 and light intensity 80 µmol/m2 s. Control plants

were kept in regular short-day conditions (16 h dark and 8 h light, red to far-red ratio = 12 and 120 µmol/m2 s light intensity) for 24 h. Light intensity and spectral composition were

measured by an Analytical Spectral Devices FieldSpec Pro spectrophotometer. For qRT-PCR analysis, total RNA was extracted from leaves or shoot apices using TRIzol (Thermo Fisher Scientific)

and purified with the RNeasy Mini Kit (Qiagen, 74104). cDNA was synthesized using SuperScript III First-Strand Synthesis (Invitrogen, 18080051) from 1 μg of RNA. Real time PCR was conducted

using a cDNA standard curve. Normalized expression levels were calculated using the 2ˆ(−delta delta CT) method with the housekeeping gene _UBQ10_ (AT4G05320) as the control. Where expression

of multiple different genes was compared, normalized gene expression is shown relative to the control treatment. Primer sequences are listed in Supplementary Table 2. YEAST ONE-HYBRID ASSAY

LFY e2 and the bZIP-binding site mutated version (e2m3) were cloned into the KpnI-HF and XhoI linearized pAbAi vector (Takara) by Gibson Assembly and integrated into the yeast genome

following the Matchmaker Gold Yeast One-Hybrid protocol (Takara) and the Y1Golden strain (Takara). Coding sequences of FD and TFL1 were cloned into the pENTRD-TOPO vector. After sequencing,

constructs were shuffled into either pDEST32 or pDEST22 (Takara) by LR reaction and transformed into the DNA-binding region containing yeast strain. Empty pDEST32 and pDEST22 served as

negative controls. Growth was assayed after serial dilution on growth media with or without 60 ng/ml Aureobasidin A (Clontech, 630499). Primer sequences are listed in Supplementary Table 2.

CHIP-QPCR, CHIP-SEQ AND DATA ANALYSIS Forty two-day-old short-day grown plants were trimmed and 1.6 g of non-bolted inflorescences were harvested from 36 plants. Chromatin

immunoprecipitation was conducted following a published protocol92 for ChIP-qPCR. For ChIP-seq, each biological replicate consisted of eight individual IP reactions pooled into one MinElute

PCR (Qiagen, 28004) purification column. For ChIP-seq and ChIP-qPCR, anti-GFP antibody (Thermo Fisher Scientific, A-11122; 1:200 dilution) and anti-GUS antibody (Abcam, ab50148; 1:200

dilution) were used. The antibodies were validated by the manufacturers. ChIP-qPCR was performed using Platinum Taq DNA Polymerase (Invitrogen, 10966034) and EvaGreen dye (Biotium, 31000).

For ChIP-qPCR, the value of the ChIP samples was normalized over that of input DNA as previously described92. Non-transgenic wild-type plants were used as the negative genetic control for

anti-GFP and anti-GUS antibody ChIP. The _TA3_ retrotransposon (AT1G37110) was used as the negative control region for ChIP-qPCR. Primer sequences are listed in Supplementary Table 2.

Anti-GFP ChIP-seq was performed for gTFL1-GFP (A), gTFL1-GFP (B), _fd-1_ gTFL1-GFP and control samples (non-epitope containing plants). Anti-GUS ChIP-seq was performed for _gFD-GUS_. Two

biological replicates were sequenced in each case. Dual index libraries were prepared for the ten ChIP samples listed above and for four input samples using the SMARTer ThruPLEX DNA-Seq Kit

(Takara Bio, R400406). Library quantification was performed with the NEBNext Library Quant Kit for Illumina (NEB, E7630). Single-end sequencing was conducted using High Output Kit v2.5

(Illumina, TG-160-2005) on the NextSeq 500 platform (Illumina). FastQC v0.11.5 was performed on both the raw and trimmed93 reads using TRIMMOMATIC v0.3693

(ILLUMINACLIP:adaptors.fasta:2:30:10 LEADING:3 TRAILING:3 MINLEN:50) to confirm sequencing quality94. Reads with MAPQ ≥ 30 (SAMtools v1.7)95 uniquely mapping to the Arabidopsis Information

Resource version 10 (TAIR 10)96 that were not flagged as PCR or optical duplicates by Bowtie2 v2.3.1(ref. 97,98) were analyzed further by principal component analysis (PCA) using the plotPCA

function of deepTools99. Reads were further processed following ENCODE guidelines100, followed by cross-correlation analysis with the predict function of MACS2 (ref. 101) to empirically

determine the fragment length. Significant ChIP peaks and summits (summit _q_ value ≤ 10−10) were identified in MACS2 for the pooled ChIP relative to the pooled negative controls (ChIP in

non-transgenic wild type). Peak overlap (≥1 bp) was computed using BEDTools intersect v2.26.0 (ref. 102) and statistical significance was computed using the hypergeometric test assuming a

‘universe’ of 10,000 possible peaks103. Heatmaps were generated using deepTools v3.1.2 (ref. 99). The 3308 TFL1 and 4422 FD peaks were mapped to 3699 and 4493 Araport11 (ref. 104) annotated

genes, respectively, if the peak summit was intragenic or located ≤4 kb upstream of the transcription start site. Recently published datasets were analyzed in identical fashion. Genomic

distribution of peak summits was called using the ChIPpeakAnno library104,105. De novo motif analysis was conducted using MEME-ChIP v5.0.2 (Discriminative Mode)106 and HOMER v4.10 (ref. 107)

for MACS2 _q_ value ≤ 10−10 peak summits (±250 base pairs) compared to genome-matched background (unbound regions from similar genomic locations as the peak summits) as previously

described108,109. GO term enrichment analyses were performed using GOSlim in agriGO v2.0 (ref. 110) and significantly enriched GO terms with _q_ value < 0.0001 (FDR, Benjamini and

Yekutieli method111) were identified. CORRELATION ANALYSES Public ChIP-seq datasets for FD30, for TFL117, LFY112 and an unpublished LFY ChIP-seq dataset from our lab (GEO accession

GSE141706) were analyzed as described above for TFL1 and FD ChIP-seq. To compare the relationship between all ChIP-seq datasets we calculated Pearson correlation coefficients for reads in

regions of interest using deeptools v3.1.299. Regions of interest were comprised of the combined significant peak regions (MACS2 ≤ _q_ value 10−10) of all ChIP-seq datasets and read signal

was derived from the sequencing-depth normalized bigwig file for each sample. RNA-SEQ AND DATA ANALYSIS A single 24 h FRP was applied to 42-day-old short-day grown _ft-10_ mutants, wild-type

and _tfl1-1_ mutants, starting at the end of the day (ZT8). After the treatment, 0.1 g of inflorescence shoots were harvested for each biological replicate after removing all leaves and

roots. Three biological replicates were prepared for each experiment. RNA quantity and quality were analyzed by Qubit BR assay (Thermo Fisher Scientific, Q10210) and Agilent RNA 6000 Nano

Kit (Agilent, 5067-1511) on an Agilent 2100 bioanalyzer, respectively. Libraries were constructed from 1 µg total RNA using the TruSeq RNA Sample Prep Kit (Illumina, RS-122-2001). After

library quantification with the NEBNext Library Quant Kit for Illumina (NEB, E7630), single-end sequencing was conducted using the NextSeq 500 platform (Illumina). RNA-seq analysis was

conducted using FastQC v0.11.5 (ref. 94) on raw sequences before and after trimming using Trimmomatic v0.36 (ref. 93) (ILLUMINACLIP:adapters.fasta:2:30:10 LEADING:3 TRAILING:3 MINLEN:50) to

confirm sequencing quality94. Reads were mapped using the STAR mapping algorithm113 (–sjdbOverhang 100 --outSAMprimaryFlag AllBestScore --outSJfilterCountTotalMin 10 5 5 5

--outSAMstrandField intronMotif --outFilterIntronMotifs RemoveNoncanonical --alignIntronMin 60 --alignIntronMax 6000 --outFilterMismatchNmax 2), to the TAIR 10 Arabidopsis genome-assembly96,

and Araport11 Arabidopsis genome-annotation104. Specific read coverage was assessed with HT-Seq114 (--stranded = ‘no’ -minaqual = 30). For PCA, raw read counts were subjected to variance

stabilizing transformation and projected into two principal components with the highest variance115,116,117. In parallel, raw reads were adjusted for library size by DESeq2 v1.24.0 (ref.

118). After PCA, two biological replicates per genotype and treatment were selected for further analysis. Gene normalized z-scores were used for k-means (MacQueen) clustering119. Pairwise

differential expression analyses were performed by comparing FRP and untreated pooled normalized read counts in each genotype using default DESeq2 parameters with no shrinkage (ref. 118) and

an adjusted _p_ value cut-off ≤ 0.005. PHOTOPERIOD SHIFT PHENOTYPE ANALYSIS A single 24-h far-red light enriched photoperiod shift (FRP) was applied to 42-day-old short-day grown _ft-10_

mutants, wild-type and _tfl1-1_ mutants as for RNA-seq, followed by further growth in short-day conditions. To asses onset of reproductive development, the number of rosette leaves formed

were counted at bolting. To analyze the inflorescence architecture, the number of sessile buds, outgrowing branches, flower branches, and single flowers subtended by a cauline leaf were

counted weekly after bolting until the first normal flower (not subtended by a cauline leaf) formed. STATISTICAL ANALYSES The Kolmogorov–Smirnov (K–S) test120 was used to assess whether the

data were normally distributed. All ChIP and qRT-PCR data were normally distributed. An unpaired one-tailed _t_-test was used to test for changes in one direction. Error bars represent the

standard error of the mean (SEM). Two to three independent biological replicates were analyzed. For multiple-group comparisons (phenotypes) the non-parametric Kruskal–Wallis test121 followed

by the Dunn’s _post hoc_ test122 were employed. Box and whisker plots display minima and maxima (whiskers), lower and upper quartile (box) and median (red vertical line). REPORTING SUMMARY

Further information on research design is available in the Nature Research Reporting Summary linked to this article. DATA AVAILABILITY The authors declare that the data supporting the

findings of this study are available within the paper, its Supplementary information files and public data repositories. Source data are provided with this paper. The ChIP-seq and RNA-seq

datasets were deposited to the GEO database (GSE141894). Individual replicates and _P_ values for all figures are provided as a source data file. Source data are provided with this paper.

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Scholar  Download references ACKNOWLEDGEMENTS We thank undergraduate students Gabriela M. Blandino, Xindi Chen and Zubaida Salman and Dietrich James Nigh for help with the experiments, Dr.

John D. Wagner for input on the manuscript and the Plant Biology group as well as Wagner lab members for feedback on this project. This research was funded by National Science Foundation IOS

grant 1557529 and 1905062 to D.W. AUTHOR INFORMATION Author notes * Cheol Woong Jeong Present address: LG Economic Research Institute, LG Twin tower, Seoul, 07336, Korea * Nobutoshi

Yamaguchi Present address: Division of Biological Science, Graduate School of Science and Technology, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara, 630-0192, Japan

* These authors contributed equally: Cheol Woong Jeong, Nobutoshi Yamaguchi. AUTHORS AND AFFILIATIONS * Department of Biology, University of Pennsylvania, 415S. University Ave, Philadelphia,

PA, 19104, USA Yang Zhu, Samantha Klasfeld, Cheol Woong Jeong, Run Jin, Nobutoshi Yamaguchi & Doris Wagner * Research Institute for Biological Sciences, Okayaka Prefecture, 7549-1,

Kibichuoh-cho, Kaga-gun, Okayama, 716-1241, Japan Koji Goto Authors * Yang Zhu View author publications You can also search for this author inPubMed Google Scholar * Samantha Klasfeld View

author publications You can also search for this author inPubMed Google Scholar * Cheol Woong Jeong View author publications You can also search for this author inPubMed Google Scholar * Run

Jin View author publications You can also search for this author inPubMed Google Scholar * Koji Goto View author publications You can also search for this author inPubMed Google Scholar *

Nobutoshi Yamaguchi View author publications You can also search for this author inPubMed Google Scholar * Doris Wagner View author publications You can also search for this author inPubMed 

Google Scholar CONTRIBUTIONS D.W. and Y.Z. conceived of the study and Y.Z. conducted the majority of the experiments. S.K. conducted the bioinformatic analyses. N.Y. and C.W.J. identified

the optimal stage to study primordium fate regulation by TFL1, conducted initial TFL1 ChIP analyses and mapped the FD-binding sites in LFY. R.J. and Y.Z. constructed and sequenced ChIP-seq

libraries, K.G generated the biologically active genomic GFP-TFL1 construct. D.W. wrote the manuscript with the help of Y.Z. and input from all other authors. CORRESPONDING AUTHOR

Correspondence to Doris Wagner. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. ADDITIONAL INFORMATION PEER REVIEW INFORMATION _Nature Communications_

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and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Zhu, Y., Klasfeld, S., Jeong, C.W. _et al._ TERMINAL FLOWER 1-FD complex target genes and competition with FLOWERING LOCUS T. _Nat

Commun_ 11, 5118 (2020). https://doi.org/10.1038/s41467-020-18782-1 Download citation * Received: 07 February 2020 * Accepted: 01 September 2020 * Published: 12 October 2020 * DOI:

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